The present invention relates to the field of recombinant DNA technology, more in particular to the field of gene therapy. Specifically, the present invention relates to gene therapy using materials derived from adenovirus, in particular human recombinant adenovirus, and relates to novel virus-derived vectors and novel packaging cell lines for vectors based on adenoviruses. Furthermore, this invention also pertains to the screening of replication-competent and revertant E1 adenoviruses from recombinant adenoviruses used in gene therapy.
Gene therapy is a recently developed concept for which a wide range of applications can be and have been envisaged. In gene therapy, a molecule carrying specific genetic information is introduced into some or all cells of a host. This results in the specific genetic information being padded to the host in a functional format. The specific genetic information added may be a gene or a derivative of a gene, such as a cDNA (which encodes a protein), or the like. In the case where cDNA is added, the encoded protein can be expressed by the machinery of the host cell.
The genetic information can also be a sequence of nucleotides complementary to a sequence of nucleotides (be it DNA or RNA) present in the host cell. With this functional format, the added DNA molecule or copies made thereof in situ are capable of base pairing with the complementary sequence present in the host cell.
Applications of such gene therapy techniques include, but are not limited to, the treatment of genetic disorders by supplementing a protein or other substance which is, through the genetic disorder, not present or at least present in insufficient amounts in the host, the treatment of tumors or other such non-acquired diseases, and the treatment of acquired diseases such as immune diseases, autoimmune diseases, infections, and the like.
As may be clear from the above, there are basically three different approaches in gene therapy. The first approach is directed toward compensating for a deficiency present in a host (such as a mammalian host). The second approach is directed toward the removal or elimination of unwanted substances (organisms or cells). The third approach is directed toward the application of a recombinant vaccine (e.g., directed against tumors or foreign micro-organisms).
Adenoviruses carrying deletions have been proposed as suitable vehicles for the purpose of gene therapy. Adenoviruses are essentially non-enveloped DNA viruses. Gene-transfer vectors derived from such adenoviruses (known as xe2x80x9cadenoviral vectorsxe2x80x9d) have several features that make them particularly useful for gene transfer. These features include, but are not limited to: 1) the fact that the biology of the adenoviruses is characterized in detail, 2) that the adenovirus is not associated with severe human pathology, 3) that the adenovirus is extremely efficient in introducing its DNA into the host cell, 4) that the adenovirus can infect a wide variety of cells and has a broad host-range, 5) that the adenovirus can be produced in large quantities with relative ease, and 6) that the adenovirus can be rendered replication defective by deletions in the early-region 1 (xe2x80x9cE1xe2x80x9d) of the viral genome, thus providing an important safety feature.
The adenovirus genome is a linear double-stranded DNA molecule of approximately 36000 base pairs with the 55 kiloDalton (xe2x80x9ckDxe2x80x9d) terminal protein covalently bound to the 5xe2x80x2 terminus of each strand. The adenovirus DNA contains identical Inverted Terminal Repeats (ITR) of about 100 base pairs with the exact length depending on the serotype. The viral origins of replication are located within the ITRs at the genome ends. The synthesis of the DNA occurs in two stages. First, the replication proceeds by strand displacement, generating a daughter duplex molecule and a parental displaced strand. The displaced strand is single stranded and can form a structure known as a xe2x80x9cpanhandlexe2x80x9d intermediate, which allows replication initiation and generation of a daughter duplex molecule. Alternatively, replication may proceed from both ends of the genome simultaneously, obviating the need to form the panhandle intermediate structure. The replication is summarized in FIG. 14 (adapted from Lechner, R. L. and Kelly Jr., T. J., xe2x80x9cThe Structure of Replicating Adenovirus 2 DNA Molecules. J. Mol. Biol. 174, pp. 493-510 (1977), hereby incorporated herein by reference).
During the productive infection cycle, the viral genes are expressed in two phases: an early phase and a late phase. The early phase is the period up to viral DNA replication, and the late phase is the period which coincides with the initiation of viral DNA replication. During the early phase, only the early gene products encoded by regions E1, E2, E3 and E4 are expressed, which carry out a number of functions that prepare the cell for synthesis of viral structural proteins (see Berk, A. J., Ann. Rev. Genet. 20, pp. 45-79 (1986), hereby incorporated herein by reference). During the late phase, the late viral gene products are expressed in addition to the early gene products and host cell DNA and protein synthesis are shut off. Consequently, the cell becomes dedicated to the production of viral DNA and of viral structural proteins (see Tooze, J., DNA Tumor Viruses (revised), Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1981), hereby incorporated herein by reference).
The E1 region of adenovirus is the first region of adenovirus expressed after infection of the target cell. This region consists of two transcriptional units, the E1A and E1B genes. Both the E1A and E1B are required for oncogenic transformation of primary (embryonal) rodent cultures. The main functions of the E1A gene products are:
i) to induce quiescent cells to enter the cell cycle and resume cellular DNA synthesis, and
ii) to transcriptionally activate the E1B gene and the other early regions (E2, E3, E4).
Transfection of primary cells with the E1A gene alone can induce unlimited proliferation (known as xe2x80x9cimmortalizationxe2x80x9d), but does not result in complete transformation. However, the expression of E1A in most cases results in the induction of programmed cell death (apoptosis), and only occasionally immortalization is obtained (see Jochemsen, et al., EMBO J. 6, pp. 3399-3405 (1987), hereby incorporated herein by reference). Co-expression of the E1B gene is required to prevent induction of apoptosis and for complete morphological transformation to occur. In established immortal cell lines, high level expression of E1A can cause complete transformation in the absence of E1B (see Roberts et al., J. Virol. 56, pp. 404-413 (1985), hereby incorporated herein by reference).
The E1B-encoded proteins assist E1A in redirecting the cellular functions to allow viral replication. The E1B 55 kD and E4 33 kD proteins, which form a complex that is essentially localized in the nucleus, function to inhibit the synthesis of host proteins and to facilitate the expression of viral genes. Their main influence is to establish selective transport of viral mRNAs from the nucleus to the cytoplasm, concomittantly with the onset of the late phase of infection. The E1B 21 kD protein is important for correct temporal control of the productive infection cycle, thereby preventing premature death of the host cell before the virus life cycle has been completed. Mutant viruses incapable of expressing the E1B 21 kD gene-product exhibit a shortened infection cycle that is accompanied by excessive degradation of host cell chromosomal DNA (deg-phenotype) and in an enhanced cytopathic effect (cyt-phenotype) (see Telling et al., xe2x80x9cAbsence of an Essential Regulatory Influence of the Adenovirus E1B 19-kiloDalton Protein on Viral Growth and Early Gene Expression in Human Diploid W138, HeLa, and A549 cells,xe2x80x9d J. Virol. 68, pp. 541-547 (1994), hereby incorporated herein by reference). The deg and cyt phenotypes are suppressed when the E1A gene is mutated, thus indicating that these phenotypes are a function of E1A (see White et al., J. Virol. 62, pp. 3445-3454 (1988), hereby incorporated herein by reference). Furthermore, the E1B 21 kD protein slows down the rate by which E1A switches on the other viral genes. It is not presently known through which mechanisms E1B 21 kD quenches these E1A dependent functions.
The vectors derived from human adenoviruses, in which at least the E1 region has been deleted and replaced by a gene of interest, have been used extensively for gene therapy experiments in the pre-clinical and clinical phase. As stated before, all adenovirus vectors currently used in gene therapy have a deletion in the E1 region, where novel genetic information can be introduced. The E1 deletion renders the recombinant virus replication defective (see Stratford-Perricaudet, L. D. and Perricaudet, M., xe2x80x9cGene Transfer into Animals: The Promise of Adenovirusxe2x80x9d, Human Gene Transfer, Cohen-Adenauer, and M. Boiron (Eds.), John Libbey Eurotext, pp. 51-61 (1991), hereby incorporated herein by reference). It has been demonstrated that recombinant adenoviruses are able to efficiently transfer recombinant genes to a rat liver and to airway epithelium of rhesus monkeys (see Bout et al., xe2x80x9cIn vivo Adenovirus-Mediated Transfer of Human CFTR cDNA to Rhesus Monkey Airway Epithelium: Efficacy, Toxicity and Safetyxe2x80x9d, Gene Therapy 1, pp. 385-394 (1994) and Bout et al., xe2x80x9cLung Gene Therapy: In Vivo Adenovirus Mediated Gene Transfer to Rhesus Monkey Airway Epitheliumxe2x80x9d, Human Gene Therapy 5, pp. 3-10 (1994), both hereby incorporated herein by reference). Additionally, researchers have observed a very efficient in vivo adenovirus mediated gene transfer to a variety of tumor cells in vitro and to solid tumors in animal models (lung tumors, glioma) and to human xenografts in immunodeficient mice (lung) in vivo (see Vincent et al., xe2x80x9cTreatment of Lepto-Meningeal Metastasis in a Rat Model Using a Recombinant Adenovirus Containing the HSV-tk Genexe2x80x9d, J. Neurosurgery in press (1996), Vincent, et al., xe2x80x9cHerpes Simplex Virus Thymidine Kinase Gene Therapy for Rat Malignant Brain Tumorsxe2x80x9d, Human Gene Therapy 7, pp. 197-205 (1996), and Blaese et al., xe2x80x9cVectors in Cancer Therapy: How Will They Deliver?xe2x80x9d, Cancer Gene Therapy 2, pp. 291-297 (1995), all of which are hereby incorporated herein by reference).
For example, in contrast to retroviruses, adenoviruses 1) do not integrate into the host cell genome, 2) are able to infect non-dividing cells, and 3) are able to efficiently transfer recombinant genes in vivo (see Brody, S. L., and Crystal, R. G., xe2x80x9cAdenovirus-Mediated In Vivo Gene Transferxe2x80x9d, Ann. N. Y. Acad. Sci. 716, pp. 90-101 (1994), hereby incorporated herein by reference). Those features make adenoviruses attractive candidates for in vivo gene transfer of, for instance, suicide or cytokine genes into tumor cells.
However, a problem associated with current recombinant adenovirus technology is the possibility of unwanted generation of replication-competent adenovirus (xe2x80x9cRCAxe2x80x9d) during the production of recombinant adenovirus (see Lochmxc3xcller et al., xe2x80x9cEmergence of Early Region 1-Containing Replication-Competent Adenovirus in Stocks of Replication-Defective Adenovirus Recombinants (DE1+DE3) During Multiple Passages in 293 Cellsxe2x80x9d, Human Gene Therapy 5, pp. 1485-1492 (1994) (hereinafter xe2x80x9cthe Lochmxc3xcller articlexe2x80x9d) and Imler et al., xe2x80x9cNovel Complementation Cell Lines Derived from Human Lung Carcinoma A549 Cells Support the Growth of E1-Deleted Adenovirus Vectorsxe2x80x9d, Gene Therapy 3, pp. 75-84 (1996), both hereby incorporated herein by reference). This is caused by homologous recombination between overlapping sequences from the recombinant vector and the adenovirus constructs present in the complementing cell line, such as the 293 cells (see Graham et al., xe2x80x9cCharacteristics of a Human Cell Line Transformed by DNA from Adenovirus Type 5xe2x80x3, J. Gen. Virol. 36, pp. 59-72 (1977) (hereinafter xe2x80x9cthe Graham articlexe2x80x9d), hereby incorporated herein by reference). RCA in batches to be used in clinical trials is undesirable because 1) RCA will replicate in an uncontrolled fashion, 2) RCA can complement replication-defective recombinant adenovirus, causing uncontrolled multiplication of the recombinant adenovirus, and 3) batches containing RCA induce significant tissue damage and hence strong pathological side effects (see the Lochmxc3xcller article). Therefore, batches to be used in clinical trials should be proven free of RCA (see Ostrove, J. M., xe2x80x9cSafety Testing Programs for Gene Therapy Viral Vectorsxe2x80x9d, Cancer Gene Therapy 1, pp. 125-131 (1994), hereby incorporated herein by reference).
As previously discussed, recombinant adenoviruses are deleted for the E1 region. The adenovirus E1 products trigger the transcription of the other early genes (E2, E3, E4), which consequently activate expression of the late virus genes. Therefore, it was generally thought that E1-deleted vectors would not express any other adenovirus genes. However, recently it has been demonstrated that some cell types are able to express adenovirus genes in the absence of E1 sequences. This indicates, that some cell types possess the machinery to drive transcription of adenovirus genes. In particular, it was demonstrated that such cells synthesize E2A and late adenovirus proteins.
In a gene therapy setting, this means that the transfer of the therapeutic recombinant gene to somatic cells not only results in expression of the therapeutic protein, but may also result in the synthesis of viral proteins. Cells that express adenoviral proteins are recognized and killed by Cytotoxic T Lymphocytes, which both eradicates the transduced cells and causes inflammations (see Bout et al., xe2x80x9cIn Vivo Adenovirus-Mediated Transfer of Human CFTR cDNA to Rhesus Monkey Airway Epithelium: Efficacy, Toxicity and Safetyxe2x80x9d, Gene Therapy 1, pp. 385-394 (1994); Engelhardt, et al., xe2x80x9cAdenovirus-Mediated Transfer of the CFTR Gene to Lung of Nonhuman Primates: Biological Efficacy Studyxe2x80x9d, Human Gene Therapy 4, pp. 759-769 (1993); and Simon et al., xe2x80x9cAdenovirus-Mediated Transfer of the CFTR Gene to Lung of Nonhuman Primates: Toxicity Studyxe2x80x9d, Human Gene Therapy 4, pp. 771-780 (1993), all of which are hereby incorporated herein by reference). As this adverse reaction is hampering gene therapy, several solutions to this problem have been suggested. These solutions include using immunosuppressive agents after treatment, retainment of the adenovirus E3 region in the recombinant vector (see patent application EP 95202213, hereby incorporated herein by reference), and using temperature sensitive (xe2x80x9ctsxe2x80x9d) mutants of human adenovirus, which have a point mutation in the E2A region (see WIPO patent application WO/28938, hereby incorporated herein by reference).
However, the strategies which circumvent the immune response have their limitations. For example, the use of ts mutant recombinant adenovirus diminishes the immune response to some extent, but was less effective in preventing pathological responses in the lungs (see Engelhardt et al., xe2x80x9cProlonged Transgene Expression in Cotton Rat Lung with Recombinant Adenoviruses Defective in E2Axe2x80x9d, Human Gene Therapy 5, pp. 1217-1229 (1994) (hereinafter xe2x80x9cthe Engelhardt 1994a articlexe2x80x9d), hereby incorporated herein by reference).
The E2A protein may induce an immune response by itself and it plays a pivotal role in the switch to the synthesis of late adenovirus proteins. Therefore, it is advantageous to make recombinant adenoviruses which are mutated in the E2 region, rendering it temperature sensitive, as has been claimed in WIPO patent application WO/28938. However, a major drawback of this system is the fact that, although the E2A protein is unstable at the non-permissive temperature, the immunogenic protein is still being synthesized. In addition, it is expected that the unstable protein activates late gene expression, albeit to a low extent. ts125 mutant recombinant adenoviruses have been tested, and prolonged recombinant gene expression has been reported (see Yang et al., xe2x80x9cInactivation of E2A in Recombinant Adenoviruses Improves the Prospect for Gene Therapy in Cystic Fibrosisxe2x80x9d, Nat. Genet. 7, pp. 362-369 (hereinafter xe2x80x9cthe Yang 1994a articlexe2x80x9d); the Engelhardt 1994a article; Engelhardt et al., xe2x80x9cAblation of E2A in Recombinant Adenoviruses Improves Transgene Persistence and Decreases Inflammatory Response in Mouse Liverxe2x80x9d, Proc. Nat""l. Acad. Sci. 91, pp. 6196-6200 (1994); Yang et al., xe2x80x9cCellular and Humoral Immune Responses to Viral Antigens Create Barriers to Lung-Directed Gene Therapy with Recombinant Adenovirusesxe2x80x9d, J. Virol. 69, pp. 2004-2015 (1995) (hereinafter xe2x80x9cthe Yang 1994b articlexe2x80x9d), all of which are hereby incorporated herein by reference). However, pathology in the lungs of cotton rats was still high (see the Engelhardt 1994a article), indicating that the use of ts mutants results in only a partial improvement in recombinant adenovirus technology. Others did not observe prolonged gene expression in mice and dogs using ts125 recombinant adenovirus (see Fang et al., xe2x80x9cLack of Persistence of E1-Recombinant Adenoviral Vectors Containing a Temperature Sensitive E2A Mutation in Immunocompetent Mice and Hemophilia Dogsxe2x80x9d, Gene Therapy 3, pp. 217-222 (1996), hereby incorporated herein by reference). An additional difficulty associated with the use of ts125 mutant adenoviruses is that a high frequency of reversion is observed. These revertants are either real revertants or the result of second site mutations (see Kruijer et al., xe2x80x9cStructure and Function of DNA Binding Proteins from Revertants of Adenovirus Type 5 Mutants with a Temperature-Sensitive DNA Replicationxe2x80x9d, Virology 124, pp. 425-433 (1983), and Nicolas et al., xe2x80x9cTemperature-Independent Revertants of Adenovirus H5ts125 and H5ts107 Mutants in the DNA Binding Protein: Isolation of a New Class of Host Range Temperature Conditional Revertantsxe2x80x9d, Virology 108, pp. 521-524 (1981), both of which are hereby incorporated herein by reference). Both types of revertants have an E2A protein that functions at normal temperature and have, therefore, similar toxicity as the wild-type virus.
E1-deleted recombinant adenovirus vectors (xe2x80x9crAVxe2x80x9d) can be propagated on dedicated helper cells. Dedicated helper cells are specialized cells that provide the E1 functions in trans, such as cell lines 293 and 911. Although encouraging results have been obtained with rAV, two major problems are associated with the use of rAVs. First, the host immune response against the adenovirus particles and the transduced cells and, second, the generation of replication-competent adenovirus (xe2x80x9cRCAxe2x80x9d) during manufacture of rAV lots. RCA include revertant vectors that reacquired the E1 region as a result of homologous recombination with E1 sequences integrated in the helper cells. An aspect of the present invention which will be described below is a new helper cell line, PER.C6(trademark), and non-overlapping E1-deleted adenoviral vectors which eliminates the problem of RCA generation by homologous recombination.
Cell line 293 has been the most frequently used cell line for the production of adenoviral vectors. This cell line was generated in the 1970s by transfection of diploid human embryonic kidney cells with sheared Adenoivrus serotype 5 (xe2x80x9cAd5xe2x80x9d) DNA in the course of a study on the transforming potential of the E1 genes of adenoviruses. Mapping of the Ad5 sequences in the genome of the 293 cells indicated the presence of contiguous Ad5 sequences from the left-hand end of the genome up to position 4137 (Evelegh et al., xe2x80x9cCloning and Sequencing of the Cellular-Viral Junctions from the Human Adenovirus Type 5 Transformed 293 Cell Linexe2x80x9d, Virology 233, pp. 423-429 (1997), hereby incorporated herein by reference). Thus, when typical E1 replacement vectors are propagated on the 293 cells, there is sequence homology between vector and helper cell DNA of up to about 450 base pairs at the left-hand side of the transgene, and about 800 base pairs at the right-hand side.
Due to this sequence overlap, the replication of rAV on the 293 cells results in the generation of RCA. This replication was first reported in the Lochmxc3xcller article wherein an E1+E3-deleted rAV was passaged multiple times on 293 cells. RCA was detected that contained E1, but lacked E3. This finding suggested that a small fraction of the rAVs had regained E1 by homologous recombination between overlapping sequences in the rAV DNA and the adenovirus DNA that is present in the 293 cells. This was later confirmed in the article by Hehir K. M. et al., xe2x80x9cMolecular Characterization of Replication-Competent Variants of Adenovirus Vectors and Genome Modifications to Prevent Their Occurrencexe2x80x9d, J. Virol. 70, pp. 8459-8467 (1996) (hereinafter xe2x80x9cthe Hehir articlexe2x80x9d), hereby incorporated herein by reference, which discloses the propagation of Ad2-based rAV on the Ad5-transformed 293 cells and the detection of RCA carrying the Ad5 E1 region, despite the presence of the entire left-hand end of the Ad5 genome in the 293 cells. All of the studied RCA isolates were found to be generated by two homologous recombination events upstream and downstream of the transgene, resulting in loss of the transgene and re-acquirement by the vector of the E1 region.
The appearance of RCA in rAV batches is a chance event and is, therefore, unpredictable and difficult to control. This is a significant problem for good manufacturing practices, particularly if large scale batches have to be prepared. A number of reports on the frequency of RCA formation during manufacture of rAVs have been published (Table 1). These data illustrate that with the conventional E1-deleted Ad5 (and adenoviruses serotype 2 (xe2x80x9cAD2xe2x80x9d)) rAVs, RCA is generated with frequencies that frustrate the large-scale production of clinical lots of rAVs.
It should be noted that homologous recombination is not the only source of RCA. During the generation of rAV, RCAs can also be introduced into the system from outside. An exemplary method of rAV construction is to co-transfect the large ClaI-fragment of Ad5 together with an adapter plasmid that carries the gene of interest into the helper cells. Incomplete restriction-enzyme digestion of the adenovirus DNA can also be responsible for RCA production (i.e., wild-type Ad5, in this example).
The use of Ad genomes cloned in bacterial plasmids eliminates this risk. In addition, inadvertent cross-contamination can occur in laboratories where replication-competent adenoviruses are propagated.
Replication-competent adenoviruses derived from rAV that are currently known are very similar to wild-type adenoviruses, except that in most cases the E3 region is deleted, which has not been observed in wild-type isolates (see the Lochmxc3xcller article and the Hehir article). Most of the rAVs used to date are derived from human adenovirus serotype 2 or 5 (i.e., Ad2 and Ad5, respectively). Ad2 and Ad5 are mainly associated with mild respiratory infections, and these viruses have a tropism mainly for epithelial cells. RCA derived from such vectors can be expected to cause disease similar to that caused by wild-type Ad5 and Ad2.
The presence of RCA in rAV batches to be used in human patients is clearly undesirable, as the RCA may replicate in an uncontrolled manner in the patient. Although the replication of the RCA is limited by the recipient""s immune system, it is a potential hazard, especially in immuno-comprised patients. In addition, RCA can rescue the vector, increasing the amount of vector shed by the patient. Rescue of the vector by RCA has been observed in cotton rats, a rodent species that is permissive for human adenovirus replication (see Imler et al., xe2x80x9cNovel Complementation Cell Lines Derived from Human Lung Carcinoma A549 Cells Support the Growth of E1-Deleted Adenovirus Vectorsxe2x80x9d, Gene Therapy 3, pp. 75-84 (1996) (hereinafter xe2x80x9cthe Imler referencexe2x80x9d), hereby incorporated herein by reference). Furthermore, the presence of RCA is associated with inflammatory responses (see Hermens et al., xe2x80x9cAdenoviral Vector-Mediated Gene Expression in the Nervous System of Immunocompetent Wistar and T Cell-Deficient Nude Rats: Preferential Survival of Transduced Astroglial Cells in Nude Ratsxe2x80x9d, Human Gene Therapy 8, pp. 1049-1063 (1997), hereby incorporated herein by reference). Such inflammatory responses may be caused by the fact that multiplication of the adenovirus causes tissue damage, or by the fact that large amounts of adenovirus proteins are synthesized that are toxic for cells (e.g., hexon and penton), and are very immunogenic. Thus, the presence of RCA in rAV batches to be used in, for example, clinical trials is undesirable, as it may induce significant pathological side effects. This is also recognized by regulatory bodies, such as the Food and Drug Administration (xe2x80x9cFDAxe2x80x9d). Therefore, labor-intensive and expensive RCA screening tests such as the tissue culture method, the supernatant rescue assay, and PCR assay are required (see Dion et al., xe2x80x9cSupernatant Rescue Assay Versus Polymerase Chain Reaction for Detection of Wild-Type Adenovirus-Contaminating Recombinant Adenovirus Stocksxe2x80x9d, J. Virol. Methods 56(1), pp. 99-107 (1996), hereby incorporated herein by reference). Although there are now options available that enable RCA-free production of rAV, screening for RCA is still required by the FDA. Screening for RCA has significantly increased the manufacturing costs of clinical rAV lots, and has led to delays in onsets of clinical studies.
Currently, intensive research efforts are focusing on the development of adenoviral vectors that have an altered tissue tropism. This is achieved by changing the genes encoding the capsid proteins, such as fiber, hexon, and penton. In these cases, the targets may be endothelium or smooth muscle cells, which are refractory to infection by wild-type Ad2 and Ad5. Thus, the presence of RCA in preparations of adenoviral vectors with altered tropism constitutes a potential safety risk. In this respect, it is noteworthy that adenoviruses with a tropism for endothelium have been shown to cause lethal infections in deer and mice (see Woods et al., xe2x80x9cSystemic Adenovirus Infection Associated with High Mortality in Mule Deer (Dolocoileus hemionus) in California, Vet. Pathol. 33(2), pp. 125-132 (1996) (hereinafter xe2x80x9cthe Woods articlexe2x80x9d) and Charles et al., xe2x80x9cMouse Adenovirus Type-1 Replication is Restricted to Vascular Endothelium in the CNS of Susceptible Strains of Micexe2x80x9d, Virology 245(2), pp. 216-228 (1998) (hereinafter xe2x80x9cthe Charles articlexe2x80x9d), both of which are hereby incorporated herein by reference). The Woods article reported on very high mortality rates in deer upon infection with adenovirus. Mortality was caused by replication of the virus in endothelium of the animal, causing severe vasculitis. In mice, mouse adenovirus (xe2x80x9cMAVxe2x80x9d) can cause lethal infections by targeting the vascular endothelium of the brain, as discussed in the Charles article. Also, in infants with an intact immune system, adenovirus infections can cause severe health problems and even death (see Munoz et al., xe2x80x9cDisseminated Adenovirus Disease in Immunocompromised and Immunocompetent Childrenxe2x80x9d, Clin. Infect. Dis. 27(5), pp. 1194-1200 (1998), hereby incorporated herein by reference). Therefore, batches of rAV with an altered tropism, to be used in clinical trials, should be free of contaminating RCA.
To reduce the immunogenicity of the rAV, and to increase the insert capacity, several groups are developing strategies to produce rAVs that are deleted of all Ad genes (so-called xe2x80x9cgutlessxe2x80x9d adenoviruses). Gutless rAVs can be propagated using a helper virus. In the most efficient system to date, an E1-deleted helper virus is used with a packaging signal that is flanked by bacteriophage P1 loxP sites (xe2x80x9cfloxedxe2x80x9d). Infection of the helper cells that express Cre recombinase with the gutless virus together with the helper virus with a floxed packaging signal should only yield gutless rAV, as the packaging signal is deleted from the DNA of the helper virus. However, if 293-based helper cells are used, the helper virus DNA can recombine with the Ad5 DNA that is integrated in the helper cell DNA. As a result, a wild-type packaging signal, as well as the E1 region, is regained. Thus, also production of gutless rAV on 293- (or 911-) based helper cells can result in the generation of RCA, if an E1-deleted helper virus is used.
Considering the magnitude of the problem, considerable research and effort has been devoted to solving the RCA problem. Strategies to circumvent RCA generation during rAV production have been focused at reducing or eliminating the sequence homology between the vector and the packaging cell line (see the Hehir article, the Imler article, and the Fallaux 1998 article). The present inventors have shown that the combination of PER.C6(trademark) helper cells (available from IntroGene of Leiden, The Netherlands) and matched vectors that do not share homologous sequences eliminates the generation of RCA by homologous recombination (see the Fallaux 1998 article). Note that in such a system, homology can also be provided by plasmid-derived sequences, as the PER.C6(trademark) cell line has been generated by transfection with a cloned adenovirus E1 region. Hehir demonstrated that deletion or relocation of the gene encoding the minor capsid protein IX resulted in a reduction of the frequency of RCA formation (see the Hehir article).
Another strategy that could prevent the formation of RCA is to delete additional essential genes from the vector backbone. Several of such strategies have been developed aiming at reducing the immunogenicity of the rAV. In most cases, rAVs are constructed with an additional deletion in the adenoviral E2 or E4 region. These rAVs are propagated on cell lines that complement both E1 as well as the other gene. Production of such rAVs on appropriate helper cell lines is expected to reduce or eliminate the risk of generating RCA, as multiple recombinations would be required. However, a potential problem associated with the use of 293-based cell lines is that homologous recombination in the E1 region of adenovirus will generate adenoviruses which have reacquired the E1 region, but still have defects in their E2 or E4 genes. Such as adenovirus revertant is not an RCA in the strict sense, as it is not able to replicate independently in human cells. However, the presence of the E1 region in such E1 revertants (designated xe2x80x9cREAxe2x80x9d: revertant E1 adenoviruses) poses another risk; that being the Ad E1 region having the potential to transform and immortalize rodent cells, and, albeit with much lower frequency, some human cell types. E1-containing adenoviruses that are deleted in either E2A or E4 are able to transform primary baby-rat kidney (BRK) cells (see Table 2). In contrast, none of the vectors that are deleted in E1 were able to transform such primary cells (see Table 2).
Ads that carry lethal deletions have in fact been shown to transform cells more efficiently than wild-type Ad5. For example, H5ts125 encodes temperature-sensitive DNA-binding proteins, due to a defect in the E2A region. This adenovirus mutant exhibits a higher transformation frequency at the non-permissive temperature than it does at the permissive temperature. It is speculated that E2- or E4-deleted Ads, in contrast to wild-type Ad, do not contain sequences that are toxic for BRK cells. Although the number of foci obtained by infection with E1-containing Ads was slightly lower compared to the amount of foci that arose upon transfection with an Ad5 E1 plasmid (see Table 2), one should bear in mind that 5xc3x97107 virus particles carry approximately 2 ng DNA, whereas present experimentation used 5 mg plasmid DNA for transfection.
Whether REAs are able to induce tumors in humans is unknown. On the one hand, given the fact that the E1A and E1B proteins contain strong CTL epitopes, the risk may be only theoretical for immunocompetent individuals. On the other hand, REAs may be harmful for immunocomprised patients.
Therefore, it is clear that there is a need to develop novel virus derived vectors and novel packaging cell lines for vectors based on adenoviruses. Furthermore, there is a need to develop methods to screen replication-competent and revertant E1 adenoviruses from recombinant adenoviruses used in gene therapy.
One embodiment of the present invention relates to a recombinant nucleic acid molecule based on or derived from an adenovirus having at least a functional encapsidating signal and at least one functional Inverted Terminal Repeat or a functional fragment or derivative thereof and having no overlapping sequences which allow for homologous recombination leading to replication-competent virus in a cell into which it is transferred. Preferably, the recombinant nucleic acid molecule is in a linear form and has an Inverted Terminal Repeat at or near both termini. Additionally, it is preferred that the linear form recombinant nucleic acid molecule be essentially in a single stranded form and have at the 3xe2x80x2 terminus a sequence complementary to an upstream part of the same strand of the nucleic acid molecule, wherein the sequence is capable of base pairing with the upstream part in a way to be able to function as a start-site for a nucleic acid polymerase, and may include all adenovirus derived genetic information necessary for replication, except for a functional encapsidation signal, preferably resulting from the action of a nucleic acid polymerase on the nucleic acid molecule. The recombinant nucleic acid of this embodiment may include functional E2A and E2B genes or functional fragments or derivatives thereof under control of an E1A independent promoter. The recombinant nucleic acid molecule may also include a host range mutation, and may further include a mutated E2 region rendering at least one of its products temperature sensitive and/or under the control of an inducible promoter. The recombinant nucleic acid molecule may, of course, by a DNA molecule. It is, of course, understood that adenovirus-like particles and packaging cells can be fabricated using the recombinant nucleic acid molecule described in this embodiment.
Another embodiment of the present invention relates to a packaging cell for packaging adenovirus derived nucleic acid molecules, wherein the packaging cell has been provided with one or more recombinant nucleic acid molecules which provide the cell with the ability to express adenoviral gene products derived from at least the E1A region and, preferably, does not have the ability to express E1B products. Preferably, the packaging cell of the present embodiment does not have the ability to express the 21 kD E1B product, which may be the result of the genetic information encoding the 21 kD E1B product not being present. The packaging cells of the present embodiment may be diploid cells, and may be of non-human origin, such as of monkey origin which, preferably, includes a host range mutated E2A region of an adenovirus.
Established cell lines (and not human diploid cells of which 293 and 911 cells are derived) are able to express E1A to high levels without undergoing apoptotic cell death, as occurs in human diploid cells that express E1A in the absence of E1B. Such cell lines are able to trans-complement E1B-defective recombinant adenoviruses, because viruses mutated for E1B 21 kD protein are able to complete viral replication even faster than wild-type adenoviruses (see Telling et. al., xe2x80x9cAbsence of an Essential Regulatory Influence of the Adenovirus E1B 19-kiloDalton Protein on Viral Growth and Early Gene Expression in Human Diploid W138, HeLa, and A549 cellsxe2x80x9d, J. Virol 68, pp. 541-547 (1994), hereby incorporated herein by reference). The constructs are described in detail below and graphically represented in FIGS. 1-5. The constructs are transfected into the different established cell lines and are selected for high expression of E1A. This is done by operatively linking a selectable marker gene (e.g., NEO gene) directly to the E1B promoter. The E1B promoter is transcriptionally activated by the E1A gene product and, therefore, resistance to the selective agent (e.g., G418 in the case NEO is used as the selection marker) results in direct selection for desired expression of the E1A gene.
Yet another embodiment of the present invention relates to a packaging cell for packaging adenovirus derived nucleic acid molecules, wherein the packaging cell has been provided with one or more recombinant nucleic acid molecules which provide the cell with the ability to express adenoviral gene products derived from at least both the E1A and the E2A region and, preferably, does not have the ability to express E1B products. The recombinant nucleic acid molecule encoding the E2A region is, preferably, under the control of an inducible promoter and/or is mutated so that at least one of its products is temperature sensitive. The packaging cell of this embodiment preferably does not have the ability to express E1B products, generally resulting from the genetic information encoding E1B products not being present. The packaging cell of this embodiment may further include the region coding for E1B and/or a marker gene, wherein the marker gene is preferably under the control of the E1B responsive promoter. Furthermore, the packaging cell of the present embodiment, preferably, does not have the ability to express the 21 kD E1B product, which may be the result of the genetic information encoding the 21 kD E1B product not being present. The packaging cells of the present embodiment may be diploid cells, and may be of non-human origin, such as of monkey origin which, preferably includes a host range mutated E2A region of an adenovirus.
A further embodiment of the present invention relates to a packaging cell harboring nucleotides 80-5788 of the human Adenovirus 5 genome. Preferably, the packaging cell line is derived from diploid human embryonic retinoblasts (HER) that harbors nt. 80-5788 of the Ad5 genome. This cell line, named 911, deposited under no. 95062101 at the ECACC, has many characteristics that make it superior to the commonly used 293 cells (see Fallaux et al., xe2x80x9cCharacterization of 911: a new helper cell line for the titration and propagation of early-region- 1-deleted adenoviral vectorsxe2x80x9d, Human Gene Therapy 7, pp. 215-222 (1996) (hereinafter xe2x80x9cthe Fallaux 1996 article), hereby incorporated herein by reference).
Still other embodiments of the present invention include a packaging cell harboring nucleotides 459-1713 of the human Adenovirus 5 genome and a packaging cell harboring nucleotides 459-3510 of the human Adenovirus 5 genome. The packaging cells of these two embodiments may be diploid cells, and may be of non-human origin, such as of monkey origin which, preferably includes a host range mutated E2A region of an adenovirus.
Yet still further embodiments of the present invention include a recombinant nucleic acid molecule based on or derived from an adenovirus, having at least a deletion of nucleotides 459-3510 of the E1 region, and a recombinant nucleic acid molecule based on or derived from an adenovirus, having a deletion of nucleotides 459-1713 of the E1 region.
Yet still another embodiment of the present invention includes a method for intracellular amplification comprising the steps of providing a cell with a linear DNA fragment to be amplified, which fragment is provided with at least a functional part or derivative of an Inverted Terminal Repeat at one terminus and providing the cell with functional E2-derived products necessary for replication of the fragment and allowing the fragment to be acted upon by a DNA polymerase. Preferably, the cell can be provided with genetic material encoding both E2A and E2B products. Most preferably, the cell can be provided with a hairpin-like structure at the terminus of the DNA fragment opposite the Inverted Terminal Repeat.
In another aspect of the present invention, the E2A coding sequences from the recombinant adenovirus genome and transfect these E2A sequences into the (packaging) cell lines containing E1 sequences to complement recombinant adenovirus vectors have been deleted.
Major hurdles in this approach are a) that E2A should be expressed to very high levels and b) that E2A protein is very toxic to cells.
The current invention in yet another aspect, therefore, discloses use of the ts125 mutant E2A gene, which produces a protein that is not able to bind DNA sequences at the non-permissive temperature. High levels of this protein may be maintained in the cells (because it is not toxic at this temperature) until the switch to the permissive temperature is made. This can be combined with placing the mutant E2A gene under the direction of an inducible promoter, such as for instance tet, methallothionein, steroid inducible promoter, retinoic acid xcex2-receptor or other inducible systems. However, in yet another aspect of the invention, the use of an inducible promoter to control the moment of production of toxic wild-type E2A is disclosed.
Two salient additional advantages of E2A-deleted recombinant adenovirus are the increased capacity to harbor heterologous sequences and the permanent selection for cells that express the mutant E2A. This second advantage relates to the high frequency of reversion of ts125 mutation. When reversion occurs in a cell line harboring ts125 E2A, this will be lethal to the cell. Therefore, there is a permanent selection for those cells that express the ts125 mutant E2A protein. Thus, one aspect of the present invention which relates to the generation of E2A-deleted recombinant adenovirus eliminates the problem of reversion in the adenoviruses.
In yet another aspect of the invention, a further improvement in the use of non-human cell lines as packaging cell lines is disclosed. For GMP production of clinical batches of recombinant viruses, it is desirable to use a cell line that has been used widely for production of other biotechnology products. Most of the latter cell lines are from monkey origin, which have been used to produce, for example, vaccines.
These cells cannot be used directly for the production of recombinant human adenovirus, as human adenovirus cannot replicate, or only replicate to low levels, in cells of monkey origin. A block in the switch of early to late phase of adenovirus lytic cycle is underlying defective replication. However, host range (xe2x80x9chrxe2x80x9d) mutations in the human adenovirus genome are described (hr 400-404) which allow replication of human viruses in monkey cells. These mutations reside in the gene encoding E2A protein (see Klessig and Grodzicker, xe2x80x9cMutations That Allow Human Ad2 and Ad5 to Express Late Genes in Monkey Cells Maps in the Viral Gene Encoding the 72 k DNA-binding Proteinxe2x80x9d, Cell 17, pp. 957-966 (1979), Klessig et al., xe2x80x9cConstruction of Human Cell Lines Which Contain and Express the Adenovirus DNA Binding Protein Gene by Cotransformation with the HSV-1 tk Genexe2x80x9d, Virus Res. 1, pp. 169-188 (1984), and Rice and Klessig, xe2x80x9cIsolation and Analysis of Adenovirus Type 5 Mutants Containing Deletions in the Gene Encoding the DNA-Binding Proteinxe2x80x9d, J. Virol. 56, pp. 767-778 (1985) (hereinafter xe2x80x9cthe Rice and Klessing articlexe2x80x9d), all of which are hereby incorporated herein by reference). Moreover, mutant viruses have been described that harbor both the hr and temperature-sensitive ts125 phenotype (see Brough et al., xe2x80x9cRestricted Changes in the Adenovirus DNA-Binding Protein that Lead to Extended Host Range or Temperature-Sensitive Phenotypesxe2x80x9d, J. Virol. 55, pp. 206-212 (1985) (hereinafter xe2x80x9cthe Brough articlexe2x80x9d), hereby incorporated herein by reference, and the Rice and Klessig article).
Therefore, the present invention includes the generation of packaging cell lines of monkey origin (e.g., VERO, CV1) that harbor:
a. E1 sequences, to allow replication of E1/E2-defective adenoviruses; and
b. E2A sequences, containing the hr mutation and the ts125 mutation, names ts400 (see the Brough article and the Rice and Klessig article) to prevent cell death by E2A overexpression; and/or
c. E2A sequences, just containing the hr mutation, under the control of an inducible promoter; and/or
d. E2A sequences, containing the hr mutation and the ts125 mutation (ts400), under the control of an inducible promoter.
Furthermore, the present invention includes:
1. Packaging constructs that are mutated or deleted for E1B 21 kD, but just express the 55 kD protein.
2. Packaging constructs to be used for generation of complementing packaging cell lines from diploid cells (not exclusively of human origin) without the need of selection with marker genes. These cells are immortalized by expression of E1A. However, in this particular case, expression of E1B is essential to prevent apoptosis induced by E1A proteins. Selection of E1-expressing cells is achieved by selection for focus formation (immortalization), as described for 293 cells (see the Graham article) and 911 cells (see the Fallaux 1996 article), that are E1-transformed human embryonic kidney (HEK) cells and human embryonic retinoblasts (HER), respectively.
3. After transfection of HER cells with construct pIG.E1B (FIG. 4), seven independent cell lines could be established. These cell lines were designated PER.C1, PER. C3, PER.C4, PER.C5, PER.C6(trademark), PER.C8 and PER.C9. PER denotes PGK-E1-Retinoblasts. These cell lines express E1A and E1B proteins, are stable (e.g., PER.C6(trademark) for more than 57 passages) and complement E1-defective adenovirus vectors. Yields of recombinant adenovirus obtained on PER cells are a little higher than obtained on 293 cells. One of these cell lines (PER.C6(trademark)) has been deposited at the ECACC under number 96022940.
4. New adenovirus vectors with extended E1 deletions (deletion nt. 459-3510). Those viral vectors lack sequences homologous to E1 sequences in the packaging cell lines. These adenoviral vectors contain pIX promoter sequences and the pIX gene, as pIX (from its natural promoter sequences) can only be expressed from the vector and not by packaging cells (see Matsui et al., Adenovirus 2 Peptide IX is Expressed Only on Replicated DNA Moleculesxe2x80x9d, Mol. Cell Biol. 6, pp. 4149-4154 (1986), hereby incorporated herein by reference, and the Imler article).
5. E2A-expressing packaging cell lines preferably based on either E1A-expressing established cell lines or E1A-E1B-expressing diploid cells. E2A expression is either under the control of an inducible promoter or the E2A ts125 mutant is driven by either an inducible or a constitutive promoter.
6. Recombinant adenovirus vectors as described before (see 4 above) but carrying an additional deletion of E2A sequences.
7. Adenovirus packaging cells from monkey origin that are able to trans-complement E1-defective recombinant adenoviruses. They are preferably co-transfected with pIG E1AE1B and pIG.NEO, and selected for NEO resistance. Such cells expressing E1A and E1B are able to transcomplement E1-defective recombinant human adenoviruses, but will do so inefficiently because of a block of the synthesis of late adenovirus proteins in cells of monkey origin (Klessig and Grodzicker, 1979). To overcome this problem, the present invention relates to generating recombinant adenoviruses that harbor a host-range mutation in the E2A gene, allowing human adenoviruses to replicate in monkey cells. Such viruses are generated as described in FIG. 12, except DNA from a hr-mutant is used for homologous recombination.
8. Adenovirus packaging cells from monkey origin as described under 7, except that they will also be co-transfected with E2A sequences harboring the hr mutation. This allows replication of human adenoviruses lacking E1 and E2A (see under 6). E2A in these cell lines is either under the control of an inducible promoter or the tsE2A mutant is used. In the latter case, the E2A gene will thus carry both the ts mutation and the hr mutation (derived from ts400). Replication-competent human adenoviruses have been described that harbor both mutations (see the Brough article and the Rice and Klessig article).
A further aspect of the invention provides otherwise improved adenovirus vectors, as well as novel strategies for generation and application of such vectors and a method for the intracellular amplification of linear DNA fragments in mammalian cells.
The so-called xe2x80x9cminimalxe2x80x9d adenovirus vectors according to the present invention retain at least a portion of the viral genome that is required for encapsidation of the genome into virus particles (the encapsidation signal), as well as at least one copy of at least a functional part or a derivative of the Inverted Terminal Repeat (ITR), that is, DNA sequences derived from the termini of the linear adenovirus genome. The vectors according to the present invention will also contain a transgene linked to a promoter sequence to govern expression of the transgene. Packaging of the so-called minimal adenovirus vector can be achieved by co-infection with a helper virus or, alternatively, with a packaging-deficient replicating helper system as described below.
Adenovirus-derived DNA fragments that can replicate in suitable cell lines and that may serve as a packaging-deficient replicating helper system are generated as follows. These DNA fragments retain at least a portion of the transcribed region of the xe2x80x9clatexe2x80x9d transcription unit of the adenovirus genome and carry deletions in at least a portion of the E1 region and deletion in at least a portion of the encapsidation signal. In addition, these DNA fragments contain at least one copy of an inverted terminal repeat (ITR). At one terminus of the transfected DNA molecule an ITR is located. The other end may contain an ITR, or alternatively, a DNA sequence that is complementary to a portion of the same strand of the DNA molecule other than the ITR. If, in the latter case, the two complementary sequences anneal, the free 3xe2x80x2-hydroxyl group of the 3xe2x80x2 terminal nucleotide of the hairpin-structure can serve as a primer for DNA synthesis by cellular and/or adenovirus-encoded DNA polymerases, resulting in conversion into a double-stranded form of at least a portion of the DNA molecule. Further replication initiating at the ITR will result in a linear double-stranded DNA molecule, that is flanked by two ITR""s, and is larger than the original transfected DNA molecule (see FIG. 13). This molecule can replicate itself in the transfected cell by virtue of the adenovirus proteins encoded by the DNA molecule and the adenoviral and cellular proteins encoded by genes in the host-cell genome. This DNA molecule cannot be encapsidated due to its large size (greater than 39000 base pairs) or due to the absence of a functional encapsidation signal. This DNA molecule is intended to serve as a helper for the production of defective adenovirus vectors in suitable cell lines.
The present invention also comprises a method for the amplification of linear DNA fragments of variable size in suitable mammalian cells. These DNA fragments contain at least one copy of the ITR at one of the termini of the fragment. The other end may contain an ITR, or alternatively, a DNA sequence that is complementary to a portion of the same strand of the DNA molecule other than the ITR. If, in the latter case, the two complementary sequences anneal, the free 3xe2x80x2-hydroxyl group of the 3xe2x80x2 terminal nucleotide of the hairpin-structure can serve as a primer for DNA synthesis by cellular and/or adenovirus-encoded DNA polymerases, resulting in conversion of the displaced strand into a double stranded form of at least a portion of the DNA molecule. Further replication initiating at the ITR will result in a linear double-stranded DNA molecule that is flanked by two ITRs and which is larger than the original transfected DNA molecule. A DNA molecule that contains ITR sequences at both ends can replicate itself in transfected cells by virtue of the presence of at least the adenovirus E2 proteins (viz. the DNA-binding protein (DBP), the adenovirus DNA polymerase (Ad-pol), and the preterminal protein (pTP). The required proteins may be expressed from adenovirus genes on the DNA molecule itself, from adenovirus E2 genes integrated in the host-cell genome, or from a replicating helper fragment, as described above.
Several groups have shown that the presence of ITR sequences at the end of DNA molecules are sufficient to generate adenovirus minichromosomes that can replicate, if the adenovirus-proteins required for replication are provided in trans, such as by infection with a helper virus (Hu et al., xe2x80x9cSymmetrical Adenovirus Minichromosomes Have Hairpin Replication Intermediatesxe2x80x9d, Gene 110, pp. 145-150 (1992) (hereinafter xe2x80x9cthe Hu articlexe2x80x9d), Wang, K., and Pearson, G. D., xe2x80x9cAdenovirus Sequences Required for Replication In Vivoxe2x80x9d, Nucl. Acids Res. 13, pp. 5173-5187 (1985), and Hay et al., xe2x80x9cReplication of Adenovirus Minichromosomesxe2x80x9d, J. Mol. Biol. 174, pp. 493-510 (1984), all of which are incorporated herein by reference). The Hu article observed the presence and replication of symmetrical adenovirus minichromosome-dimers after transfection of plasmids containing a single ITR. The authors were able to demonstrate that these dimeric minichromosomes arise after tail-to-tail ligation of the single ITR DNA molecules. In DNA extracted from defective adenovirus type 2 particles, dimeric molecules of various sizes have also been observed using electron-microscopy (see Daniell, E. xe2x80x9cGenome Structure of Incomplete Particles of Adenovirusxe2x80x9d, J. Virol. 19, pp. 685-708 (1976) (hereinafter xe2x80x9cthe Daniell article), hereby incorporated herein by reference). It was suggested that the incomplete genomes were formed by illegitimate recombination between different molecules and that variations in the position of the sequence at which the illegitimate base pairing occurred were responsible for the heterogeneous nature of the incomplete genomes. Based on this mechanism it was speculated that, in theory, defective molecules with a total length of up to two times the normal genome could be generated. Such molecules could contain duplicated sequences from either end of the genome. However, no DNA molecules larger than the full-length virus were found packaged in the defective particles (see the Daniell article). This can be explained by the size-limitations that apply to the packaging. In addition, it was observed that in the virus particles DNA-molecules with a duplicated left-end predominated over those containing the right-end terminus (see the Daniell article). This is fully explained by the presence of the encapsidation signal near that left-end of the genome (see Grxc3xa4ble, M., and Hearing, P., xe2x80x9cAdenovirus Type 5 Packaging Domain is Composed of a Repeated Element That is Functionally Redundantxe2x80x9d, J. Virol. 64, pp. 2047-2056 (1990); Grxc3xa4ble, M., and Hearing, P., xe2x80x9ccis and trans Requirements for the Selective Packaging of Adenovirus Type-5 DNAxe2x80x9d, J Virol 66, pp. 723-31 (1992); and Hearing et al., xe2x80x9cIdentification of a Repeated Sequence Element Required for Efficient Encapsidation of the Adenovirus Type 5 Chromosomexe2x80x9d, J. Virol. 61, pp. 2555-2558 (1987), all of which are hereby incorporated herein by reference).
The major problems associated with the current adenovirus-derived vectors are:
A) The strong immunogenicity of the virus particle.
B) The expression of adenovirus genes that reside in the adenoviral vectors, resulting in a Cytotoxic T-cell response against the transduced cells.
C) The low amount of heterologous sequences that can be accommodated in the current vectors (up to maximum of approximately 8000 bp. of heterologous DNA).
Ad A) The strong immunogenicity of the adenovirus particle results in an immunological response of the host, even after a single administration of the adenoviral vector. As a result of the development of neutralizing antibodies, a subsequent administration of the virus will be less effective or even completely ineffective. However, a prolonged or persistent expression of the transferred genes will reduce the number of administrations required and may bypass the problem.
Ad B) Experiments performed by Wilson and collaborators (see U.S. Pat. No. 5,652,224) have demonstrated that after adenovirus-mediated gene transfer into immunocompetent animals, the expression of the transgene gradually decreases and disappears approximately 2-4 weeks post-infection (see the Yang 1994a article and the Yang 1994b article). This is caused by the development of a Cytotoxic T-Cell (CTL) response against the transduced cells. The CTLs were directed against adenovirus proteins expressed by the viral vectors. In the transduced cells synthesis of the adenovirus DNA-binding protein (the E2A-gene product), penton and fiber proteins (late-gene products) could be established. These adenovirus proteins, encoded by the viral vector, were expressed despite deletion of the E1 region. This demonstrates that deletion of the E1 region is not sufficient to completely prevent expression of the viral genes (see the Englehardt 1994a article).
Ad C) Studies by Graham and collaborators have demonstrated that adenoviruses are capable of encapsidating DNA of up to 105% of the normal genome size (see Bett et al., xe2x80x9cPackaging Capacity and Stability of Human Adenovirus Type-5 Vectorsxe2x80x9d, J. Virol. 67, pp. 5911-5921 (1993), hereby incorporated herein by reference). Larger genomes tend to be unstable resulting in loss of DNA sequences during propagation of the virus. Combining deletions in the E1 and E3 regions of the viral genomes increases the maximum size of the foreign DNA that can be encapsidated to approx. 8.3 kb. In addition, some sequences of the E4 region appear to be dispensable for virus growth (adding another 1.8 kb to the maximum encapsidation capacity). Also the E2A region can be deleted from the vector, when the E2A gene product is provided in trans in the encapsidation cell line, adding another 1.6 kb. It is, however, unlikely that the maximum capacity of foreign DNA can be significantly increased further than 12 kb.
Thus, the present invention includes a new strategy for the generation and production of helper-free stocks of recombinant adenovirus vectors that can accommodate up to 38 kb of foreign DNA. Only two functional ITR sequences, and sequences that can function as an encapsidation signal need to be part of the vector genome. Such vectors are called minimal adenovectors. The helper functions for the minimal adenovectors are provided in trans by encapsidation-defective replication-competent DNA molecules that contain all the viral genes encoding the required gene products, with the exception of those genes that are present in the host-cell genome, or genes that reside in the vector genome.
With the development of new generations of rAVs, the RCA problem has become more complex using conventional cell lines like 293 and 911 because a rAV revertant can be the classical RCA (i.e., which lost the transgene, regained E1, and is replication-competent), or revertant E1 adenoviruses (xe2x80x9cREAxe2x80x9d) (i.e., reacquired E1, but is still replication-defective). Thus, the present invention further involves screening rAV lots, especially those intended for clinical use, for the presence of adenovirus E1 sequences, as this will reveal RCAs, as well as REAs. Further, the present invention involves employing vector systems that prevent the formation of RCA and/or REA. Currently, adenoviral vectors are the most efficient vectors for gene-therapy applications. Adenoviral vectors are, therefore, being manipulated extensively to make them suitable for specific applications. Such developments should be accompanied by the parallel development of procedures to make rAV a safe pharmaceutical product: a manufacturing process that prevents contamination of the viral preparations with either RCA or replication-defective revertants. Despite the fact that no accidents have happened so far with RCA-contaminated rAV preparations in clinical trials, for improving the Ad vector system for gene therapy purposes, therapeutic potential and safety should be enhanced. The use of PER.C6(trademark) cells and non-overlapping vectors eliminates this problem, and allows production of safe clinical grade batches of rAVs. Only safe production systems, developed in parallel with appropriate testing methods, will warrant safe clinical application of rAVs. It is also an aspect of the present invention to molecularly characterize the revertants that are generated in the newer helper/vector combinations.